Confocal optical scanning device

ABSTRACT

The invention concerns a confocal optical scanning device comprising a mobile scanning mirror, reflection on one side  101 ( b ) compensating reflection on the opposite side  101 ( a ) after passing through an array of microscopic holes. The invention is applicable to rapid 3D and 2D microscopy, in biology and in the study of materials.

The invention concerns an optical scanning device intended to simultaneously scan an observed plane and an image plane.

Patent number PCT/FR01/02890 describes a scanning device intended to simultaneously scan an observed plane and an image plane.

Two types of scanning device are described in this document:

-   devices using an array of micromirrors for filtering the light beam     and using only one face of a movable mirror, -   devices using one or more microscopic holes as a spatial filtering     device, in order to filter the light beam, and using the two     opposite faces of a movable mirror.

The scanning devices in the second category use a movable mirror having:

-   an object face directed towards the observed object (and therefore     towards the microscope lens), which makes it possible to move, in     the plane of the array of microscopic holes, the geometric image of     a fixed point of the observed object, -   an image face directed towards the detection device (for example the     camera) which makes it possible to bring the geometrical image of a     fixed point of the observed image back to a fixed point of the     detection device.

The object face therefore allows for the scanning itself, and the image face allows compensation of this scanning.

FIGS. 1 to 5 give a better understanding of the compensation mechanism. FIG. 1(a) shows a notional configuration having simple symmetry properties. The beam arriving horizontally on the object face 01 is reflected upwards. The beam arriving vertically on the image face 02 is reflected to the right. FIG. 1(b) shows the scanning and compensation principle used in the prior art. The beam arrives horizontally on the object face and is reflected upwards. The beam arrives horizontally on the image face and is reflected downwards.

If the movable mirror undergoes a rotation about an axis orthogonal to the plane of the figure, as indicated in FIG. 2, the reflected beams in FIG. 1(a) each undergo a rotation in the direction indicated by FIG. 2(a). By reversing the direction of the beams, there is derived therefrom the direction which the incident beams must have on the image face of the movable mirror, in the scanning and compensation system of FIG. 1(b), for the direction of the output beam to remain constant. This direction is shown in FIG. 2(b). The lens and mirror system restoring the light beam of the object face to the image face of the movable mirror must therefore be designed so that the beam reflected by the object face and diverted towards the right of the figure reaches the image face whilst being diverted upwards.

If the movable mirror undergoes a small rotation about an axis situated in the plane of the figure, as indicated in FIG. 3, the directions of the beams in FIGS. 2(a) and 2(b) are modified as indicated respectively in FIGS. 3(a) and 3(b). In these figures, a cross represents a beam moving away from the observer, and a dot in a circle represents a beam moving towards the observer.

However, the situation depicted in FIG. 3 is valid only to the first order. When the angles of rotation of the movable mirror become sufficiently large, a second-order phenomenon appears, which is a rotation to the right of the beam reflected by the object face. The diagrams in FIG. 3 must then be modified as indicated in FIG. 4.

So that compensation takes place correctly to the first order, the system of lenses and mirrors which brings the beam back from the object face towards the image face must make it possible to verify the situations in FIGS. 2(b) and 3(b). Consequently, a beam reflected by the object face as indicated in FIG. 4(a) returns to the image face as indicated in FIG. 5. However in order to start off again in a constant direction it should return as indicated in FIG. 4(b). There is an incompatibility between the first-order compensation of all the movements of the mirror and the second-order compensation of the rotation movements about an axis situated in the plane of the figure. The system being designed so that the compensation takes place correctly to the first order, the second-order deviation is not compensated for but amplified, as indicated in FIG. 5. Consequently the image obtained on the detector is affected by a second-order disturbance with respect to the magnitude of the rotary movements of the mirror about an axis situated in the plane of the figure.

According to this invention, this problem is resolved by means of a confocal optical scanning device comprising:

-   at least one rotationally movable mirror comprising an object face,     for redirecting a light beam coming from an observed object, -   at least two control lenses through which the light beam passes     between the object face and an image face of the movable mirror,     opposite to the object face, -   at least one microscopic hole placed in an intermediate image plane     between two control lenses, -   redirection mirrors redirecting the light beam coming from the     observed object and having been reflected by the object face,     towards the image face of the movable mirror, in order to allow the     return of the beam to the image face, and so that a beam incident in     a fixed direction on the object face leaves the image face with a     fixed direction, independent of the position of the movable mirror,     for a set of useful rotation angles of the movable mirror,

said redirection mirrors and said control lenses being disposed so that a beam incident on the image face coming from the object face has the same axis and the same direction as the same beam when it leaves the object face in the direction of the image face.

The problem is thus resolved by means of a scanning system in which the beam incident on the object face is braught back the image face in the manner indicated in FIG. 6(a), namely that the beam reflected by the object face returns to the image face with the same axis and the same direction as just after its reflection by the object face, and the beam leaving the movable mirror after reflection on the image face has the same axis and the same direction as the incident beam on the movable mirror before reflection on the object face. The compensation for the movements of the movable mirror then takes place as indicated in FIGS. 6(b) and (c) and there is no incompatibility between the first-order compensation and the second-order compensation. The system of lenses and mirrors making it possible to bring the beam reflected by the object face back towards the image face, in the manner indicated by FIG. 6(c), also brings the beam reflected by the object face of FIG. 6(b) back towards the image face, as indicated by FIG. 6(b). In general terms it is possible to show that the compensation is perfect at all orders.

In the case where the movable mirror has two distinct axes of rotation, this configuration makes it possible to use high angles of rotation about two axes. In the case where the movable mirror has only one axis of rotation, this configuration improves the robustness of the system with respect to the imprecisions in positioning of the mirror and allows the use of the most practical axis of rotation having regard to the space requirements. In general terms, this configuration makes it possible to enlarge the useful angles of rotation of the movable mirror. However, these angles of rotation remain limited by the aberrations of the lenses and by the fact that the beam must not depart from a trajectory causing it to pass through the control lenses, the redirection mirrors and the array of microscopic holes.

This configuration also makes it possible to resolve the problem of vibrations which could be transmitted from the scanning system to the microscope lens and/or the observed sample. To this end, and according to one characteristic of the invention, the scanning device is also characterized by the following facts:

-   it comprises at least one object lens through which there passes the     light coming from the observed object and directed towards the     object face of the movable mirror, -   it comprises at least one image lens through which there passes the     light coming from the observed object and having previously been     reflected successively by the object and image faces of the movable     mirror, and directed towards a plane where the image of the observed     object forms, -   the object and image lenses are fixed to a first frame, -   the movable mirror, the control lenses and the redirection mirrors     are fixed to a second frame, -   the first and second frames can move with respect to each other and     are connected to each other only by deformable links.

The deformable links can typically be springs or rubber elements. The assembly consisting of the movable mirror and the system of mirrors and lenses restoring the beam from the object face to the image face then constitutes a whole fixed and suspended so as to isolate it mechanically from the rest of the microscopy device (object lens and image lens). In the absence of an array of microscopic holes, movements of this assembly have very little influence on the quality of the observed image. When there is an array of microscopic holes, this influence remains small provided that any oscillations are at a frequency sufficiently lower than the scanning frequency. This is because:

-   the passage of the light beam from an element linked to the first     frame to an element linked to the second frame, movable with respect     to the first, takes place in an afocal area, which avoids any     influence on the focusing plane of the translation movements of the     second frame with respect to the first, -   in a reference frame linked to the second frame, the direction of     the beam leaving the image face is the same as the direction of the     beam reaching the object face, whatever the direction of the     incident beam on the object face. This remains true in a reference     frame relating to the first frame, and whatever the movements of the     second frame with respect to the first the direction of the beam     leaving the image face remains the same as the direction of the     incident beam on the object face. This implies that the image in the     image plane (after passing from the image lens) of a fixed point of     the observed object remains fixed when the second frame moves in     rotation with respect to the first.

According to one characteristic of the invention, a simple solution applicable when the vibrations are not too great in the environment consists of the first frame being placed on a vibration-damping table, and in that the second frame is directly connected to the floor. The deformable link consists, in this case, of the suspension system of the vibration-damping table. If the vibrations are too great, the first frame is placed on a first vibration-damping table and the second frame is placed on a second independent vibration-damping table independent of the first.

The invention also makes it possible to resolve the problem of space requirement encountered when it is sought to insert the system in the optical path of an existing microscope. This is because, in this case, the movable mirror must be inserted in the afocal part of the optical path without this insertion requiring a modification to this path (change in direction for example) and without the system excessively extending the optical path. The configuration adopted makes it possible to preserve the direction of the optical path when passing through the device, and to limit the space requirement, on the optical path, to the width of the movable mirror (the remainder of the scanning device being for example on the side).

Such a scanning device can be produced in various ways. One aim of the invention is to permit the production of such a device whilst minimizing the space requirement and optimizing its transmissivity.

This aim of the invention is achieved by means of a scanning device characterized by the following facts:

-   it comprises exactly two groups of control lenses, -   each of these groups separates an afocal area in which one face of     the movable mirror is situated, from a focusing area in which an     array of microscopic holes is situated, -   it comprises exactly 4 redirection mirrors.

This is because a scanning device according to the invention contains at a minimum two lenses, in order to pass from the afocal area where the object face of the movable mirror is situated to the image plane where the array of microscopic holes is situated, and conversely to pass from the array of microscopic holes to the afocal area where the image face of the movable mirror is situated. Moreover, it can be shown that it is not possible, using exactly two lenses, to bring the beam issuing from the object face of the movable mirror back towards the image face of the movable mirror, and to obtain a compensation effect, using less than 4 mirrors. The configuration with 4 mirrors and two lenses is therefore the most optimum.

However, using 4 mirrors and 2 lenses does not for all that guarantee correct compensation by the image face of the scanning carried out by the object face. For this compensation to be effective, and according to one characteristic of the invention, the device must satisfy the following condition: (AB×BC,BC×CD)_(BC)+(BC×CD,CD×DE)_(CD)+(CD×DE,DE×EF)_(DE)+(DE×EF,EF×BC)_(EF)=π where (UV×VX,VX×XY)_(VX) represents the angle between the vector product (UV×VX) of the vectors UV and VX, and the vector product (VX×XY) of the vectors VX and XY, oriented by the vector VX, for any set of points U,V,X,Y, and where:

-   A is the point on the object face of the movable mirror which is     situated on the optical axis, -   B is the point on the first redirection mirror reached by a beam     directed from the object face to the image face of the movable     mirror, which is situated on the optical axis, -   C is the point on the second redirection mirror reached by a beam     directed from the object face to the image face of the movable     mirror, which is situated on the optical axis, -   D is the point on the third redirection mirror reached by a beam     directed from the object face to the image face of the movable     mirror, which is situated on the optical axis, -   E is the point on the fourth redirection mirror reached by a beam     directed from the object face to the image face of the movable     mirror, which is situated on the optical axis, -   F is the point on the image face of the movable mirror which is     situated on the optical axis.

This condition implies in particular that the points A, B, C, D, E, F are non-coplanar.

QUICK DESCRIPTION OF THE FIGURES

FIGS. 1 to 5 serve to support the explanation of a default of the microscopes according to the state of the art. FIGS. 1(a), 2(a), 3(a), 4(a) show a symmetrical configuration making it possible, by reversing the direction of the beam, to obtain FIGS. 1(b), 2(b), 3(b), 4(b), which illustrate the compensation principle used in the state of the art. FIG. 1 shows the central portion of the movable mirror. FIG. 2 shows the effect of a rotation movement about an axis orthogonal to the plane of the figure. FIG. 3 shows the first-order effect of a rotation movement about an axis situated in the plane of the figure. FIG. 4 shows the second-order effect of this movement. FIG. 5 shows the second-order effect of this movement when the system is configured so as to compensate for the rotations of the movable mirror to the first order.

FIG. 6 shows the principle used according to the invention for effecting the compensation of the scanning movement. FIG. 6(a) shows a central position of the movable mirror. FIG. 6(b) shows the effect of a rotation about an axis orthogonal to the plane of the Figure. FIG. 6(c) shows the effect of a rotation about an axis situated in the plane of the figure.

FIG. 7 shows a first example embodiment of the invention. FIG. 8 shows the optical path used in FIG. 7 but without the folding due to the redirection mirrors. FIG. 9 shows the principle of redirection by the mirrors and defines the points A, B, C, D, E, F characterizing this redirection.

FIGS. 10 to 15 relate to a second embodiment of the invention. FIG. 10 shows the “unfolded” optical path without the redirection mirrors. FIG. 11 shows an example of a position in 3D space of the points A, B, C, D, E, F which define the folding.

FIGS. 12 to 15 relate to a particular example of this second embodiment, completely dimensioned. FIG. 12 shows a lens used, composed of two identical achromatic lenses. FIGS. 13 to 15 show several views of the device, FIG. 15 being a perspective view.

FIRST EMBODIMENT

This first embodiment is depicted in FIG. 7. A light beam coming from an image plane 112 of a microscope, not shown, passes through an “object” lens 111, one focal plane of which is the plane 112. The figure depicts the beam coming from a particular point on the observed object. After passing through the lens 111 the beam is in an afocal area, that is to say the beam coming from a given point on the plane 112 becomes parallel after passing through the lens 111. The beam then reaches the scanning and compensation assembly 120, the entry and exit of which are in an afocal area. The first element encountered by the beam in the scanning device is the object face 101(a) of the galvanometric mirror situated in a focal plane of the lens 111. This face of the galvanometric mirror reflects the beam towards the lens 102, one focal plane of which is on the face 101(a) of the galvanometric mirror. After passing through the lens 102 the beam reaches the mirror 103, which reflects the beam towards the array of microscopic holes 104 situated in a focal plane of the lens 102. The beam, having passed through the array of microscopic holes 104, is then reflected by the mirror 105 and then passes through the lens 106, one focal plane of which is on the array 104. It then passes through the lens 107, one focal plane of which is merged with a second focal plane of the lens 106. It is reflected by the mirrors 108 and 109 and then passes through the lens 110, one focal plane of which is merged with the second focal plane of the lens 107. It is reflected by the image face 101(b) of the galvanometric mirror and leaves the scanning device 120. It is then focused in a image plane 114 by the “image” lens 113. Typically, a CCD sensor is placed in the plane 114. The lenses 102, 106, 107, 110 are identical to each other. The lighting beam directed towards the observed object can be injected at various points, in the scanning device or outside this device. The case is depicted here where a beam 115 issuing from a mercury vapor lamp is injected into the system by means of a dichroic mirror 100. The arrows depict only the direction of the beam coming from the observed object; the lighting beam being directed towards the object in the opposite direction. It is verified that in this embodiment the compensation for the scanning is effective:

-   the direction of the beam coming from the observed object is the     same before reflection on the object face of the galvanometric     mirror and after reflection on the image face of the galvanometric     mirror, -   consequently the geometric image of a fixed point on the plane 112     is a fixed point on the plane 114.

FIG. 8 illustrates the optical path of the scanning and compensation assembly in this embodiment, according to an “unfolded” representation not taking into account the mirrors. The beam issuing from the object face 101(a) of the galvanometric mirror passes successively through the lens 102, the array of microscopic holes 104, the lens 106, the lens 107 and the lens 110, and returns to the image face 101(b) of the galvanometric mirror. The diagram in FIG. 7 is obtained by folding this optical path by means of the redirection mirrors 103, 105, 108, 109. The positions of these mirrors and therefore the characteristics of the folding of the optical path can be characterized by the coordinates of the points of these mirrors which are situated on the optical axis. These mirrors and points have been depicted in FIG. 9. Point A is the centre of the object face 101(a) of the galvanometric mirror, situated on the optical axis 121. Point B is the point on the mirror 103 which is on the optical axis 121. Likewise, points C, D, E, F are the points on the mirrors successively reached by the beam and situated on the optical axis. In the present case the points A, B, C, D, E, F are coplanar.

The system can be divided into two subassemblies 440 and 441, which may be mechanically isolated from each other. For example, the subassembly 441 can be connected to a vibration-damping table, and the subassembly 440 can be connected directly to the floor. Two subassemblies can also be connected to each other by rubber parts cutting out the high vibration frequencies, the assembly being placed on a vibration-damping table. This mechanical isolation of the two subassemblies prevents transmission of vibration from the galvanometric mirror which would interfere with the functioning of the interferometric device. This mechanical isolation is made possible by the optical configuration of the scanning system: the incident beam on the face (a) of the galvanometric mirror with a given direction regains exactly the same direction after reflection on the face (b), even if the assembly 440 has moved or turned with respect to the assembly 441. Consequently the relative movements of the two assemblies, as long as they are of small magnitude, do not modify the position of the image of a point on the object 401 on the camera 430. Consequently, relative movements of the two subassemblies can be tolerated, which makes it possible to isolate them on a vibratory level.

Second Embodiment

This second embodiment consists of using not 4 lenses separating afocal areas and image planes, as in the previous case, but only two lenses or groups of lenses each separating an afocal area from an image plane.

FIG. 10 shows the “unfolded” representation of this embodiment, according to the same principle as FIG. 8. The beam reflected by the object face 201(a) of the galvanometric mirror passes through the lens L1, the array of microscopic holes 204 and the lens L2, and arrives at the image face 201(b) of the galvanometric mirror. The lenses L1 and L2 are identical and have in each case a focal plane on the array 204 and a focal plane on one of the faces of the galvanometric mirror. The optical path is folded by means of 4 mirrors as in the previous case. However, a simple folding configuration like the one illustrated in FIG. 9 is not suitable since it does not allow the return of the beam to the galvanometric mirror in the orientation illustrated by FIGS. 6(b) and 6(c), which is essential to correct compensation.

For there to be correct compensation of the scanning in this case, the folding must be carried out in a more appropriate manner. The condition which must be satisfied for the folding to take place correctly is as follows: (AB×BC,BC×CD)_(BC)+(BC×CD,CD×DE)_(CD)+(CD×DE,DE×EF)_(DE)+(DE×EF,EF×BC)_(EF)=π where for example (AB×BC,BC×CD)_(BC) represents the angle between the vector products (AB×BC) and (BC×CD), oriented by the vector BC.

This condition cannot be satisfied if the points A, B, C, D, E, F are coplanar. It is therefore necessary to use an optical path in which the elements of the scanning device are not all in the same plane. For example, the path ABCDEF can be of the type illustrated in FIG. 11, in several views, with an indication of reference frames. In general terms, this path is characterized by the three-dimensional coordinates of the points A, B, C, D, E, F. The orientations of the mirrors are derived therefrom. For example, the mirror passing through the point B is oriented so that the normal to the mirror is the bisector of the angle (BA, BC). The other mirrors are likewise oriented so that the optical axis does indeed fold along the path ABCDEF. A system satisfying this condition can be calculated, for example using the “target value” function of a spreadsheet to adjust the position of the points so as to obtain the appropriate value of the sum of angles hereinabove whilst maintaining the total length of the optical path at the value imposed by a choice of lenses.

A particular example is shown in FIGS. 12 to 15. FIG. 12 illustrates the lens used. It is composed of two identical achromatic lenses. The points P and Q are used to characterize the position of the lens. Lenses L1 and L2 being identical, the point P will be denoted P1, P2 depending on whether it is a case of the lens L1 or L2, and likewise the point Q will be denoted Q1, Q2 depending on whether it is a case of the lens L1 or L2. The characteristics of this lens are: Glass V1 SF10; index 1.73366143 (at 550 nm) Glass V2 BAK4, index n = 1.57099293 (at 550 nm) R1 483.71 mm d1 4.1 mm R2 148.27 mm d2 9 mm R3 −194.96 mm d3 12 mm PQ 38.2 mm

The thickness of the galvanometric mirror used is 6 mm. FIGS. 13 to 15 illustrate the optical path of the beam in different views. The configuration is characterized by the coordinates of the points A, B, C, D, E, F in an orthonormal reference frame, in mm: Point/ coor- di- nate A B C D E F x 0.0000 219.9341 219.9341 −39.2426 −39.2426 −4.2426 y 0.0000 0.0000 68.7576 4.2426 4.2426 4.2426 z 0.0000 0.0000 13.1298 45.0000 0.0000 0.0000 The equation: (AB×BC,BC×CD)_(BC)+(BC×CD,CD×DE)_(CD)+(CD×DE,DE×EF)_(DE)+(DE×EF,EF×BC)_(EF)=π is satisfied.

The distances along the optical axis are as follows in nun, the letter R designating the position of the array of microscopic holes 204: AP1 142.881 P1Q1 38.2 Q1B 38.852 BC 70 CR 29.523 RQ2 138.375 Q2P2 38.2 P2D 62.882 DE 45 EF 35 FIGS. 13 to 15 illustrate this example embodiment in several views. In the perspective view of FIG. 15, only the folding points A, B, C, D, E, F have been indicated. In the other two views all the points in the above table have been indicated. The object and image lenses (the equivalents of the lenses 111 and 114 of FIG. 7) have not been shown and therefore only the scanning and compensation assembly has been shown, equivalent to the assembly 120 in FIG. 7. In the complete scanning device, the object and image lenses must be added in the same way as in FIG. 7. The scanning and compensation assembly depicted in FIGS. 13 and 15 can be fixed to a single frame directly connected to the floor, the object and image lenses then being fixed to the same optical table as the microscope used.

INDUSTRIAL APPLICATIONS

The present scanning device can be used for confocal scanning microscopy, for example for cell biology. 

1. A confocal optical scanning device comprising: at least one rotationally movable mirror comprising an object face, for redirecting a light beam coming from an observed object, at least two control lenses through which the light beam passes between the object face and an image face of the movable mirror, opposite to the object face, at least one microscopic hole placed in an intermediate image plane between two control lenses, redirection mirrors redirecting the light beam coming from the observed object and having been reflected by the object face, towards the image face of the movable mirror, in order to allow the return of the beam to the image face, and so that a beam incident in a fixed direction on the object face leaves the image face with a fixed direction, independent of the position of the movable mirror, for a set of useful rotation angles of the movable mirror, said redirection mirrors and said control lenses being disposed so that a beam incident on the image face coming from the object face has the same axis and the same direction as the same beam when it leaves the object face in the direction of the image face.
 2. A scanning device as claimed in claim 1, wherein: it comprises at least one object lens through which there passes the light coming from the observed object and directed towards the object face of the movable mirror, it comprises at least one image lens through which there passes the light coming from the observed object and having previously been reflected successively by the object and image faces of the movable mirror, and directed towards a plane where the image of the observed object forms, the object and image lenses are fixed to a first frame, the movable mirror, the control lenses and the redirection mirrors are fixed to a second frame, the first and second frames can move with respect to each other and are connected to each other only by deformable links.
 3. A scanning device as claimed in claim 2, wherein the first frame is placed on a vibration-damping table and the second frame is directly connected to the floor.
 4. A scanning device as claimed in claim 2, wherein the first frame is placed on a first vibration-damping table and the second frame is placed on a second vibration-damping table independent of the first.
 5. A scanning device as claimed in one of claims 1 to 4, wherein: it comprises exactly two groups of control lenses, each of these groups separates an afocal area in which one face of the movable mirror is situated, from a focusing area in which an array of microscopic holes is situated, it comprises exactly 4 redirection mirrors.
 6. A scanning device as claimed in claim 5, satisfying the condition (AB×BC,BC×CD)_(BC)+(BC×CD,CD×DE)_(CD)+(CD×DE,DE×EF)_(DE)+(DE×EF,EF×BC) _(EF)=π where (UV×VX,VX×XY)_(VX) represents the angle between the vector product (UV×VX) of the vectors UV and VX, and the vector product (VX×XY) of the vectors VX and XY, oriented by the vector VX, for any set of points U,V,X,Y, and where: A is the point on the object face of the movable mirror which is situated on the optical axis, B is the point on the first redirection mirror reached by a beam directed from the object face to the image face of the movable mirror, which is situated on the optical axis, C is the point on the second redirection mirror reached by a beam directed from the object face to the image face of the movable mirror, which is situated on the optical axis, D is the point on the third redirection mirror reached by a beam directed from the object face to the image face of the movable mirror, which is situated on the optical axis, E is the point on the fourth redirection mirror reached by a beam directed from the object face to the image face of the movable mirror, which is situated on the optical axis, F is the point on the image face of the movable mirror which is situated on the optical axis. 